CN101928348A

CN101928348A - Human TSHR extracellular fusion protein and preparation method thereof

Info

Publication number: CN101928348A
Application number: CN2010101038703A
Authority: CN
Inventors: 施秉银; 田竹芳; 杨琪; 伍丽萍; 徐利; 高雷
Original assignee: First Affiliated Hospital of Medical College of Xian Jiaotong University
Current assignee: First Affiliated Hospital of Medical College of Xian Jiaotong University
Priority date: 2010-02-01
Filing date: 2010-02-01
Publication date: 2010-12-29

Abstract

The invention discloses human TSHR extracellular fusion protein and a preparation method thereof. The N end of the human TSHR extracellular fusion protein is connected with exogenous secretion signal peptide, and the C end thereof is connected with a purification marker sequence; when the fusion protein is expressed and purified, the fusion protein the N end of which is connected with the exogenous secretion signal peptide is resected, and the specific amino acid sequence is shown in SEQ ID No.1. The invention obtains high-expression soluble recombinant protein with the antigenic secretion form of TSH receptor in a good in-vitro expression system by constructing recombinant pichia pastoris containing pPICZ alpha A-hTSHRecd expression vector.

Description

A kind of human TSHR extracellular fusion protein and preparation method thereof

Technical field

The invention belongs to biological technical field, relate to reorganization and biotechnology preparation, particularly a kind of human TSHR extracellular fusion protein and preparation method thereof of people's tsh receptor.

Background technology

Autoimmune thyroid disease (Autoimmune thyroid disease, AITD) belong to the organ specificity disease, comprise the Graves disease (Graves ' disease, GD), struma lymphomatosa (Hashimoto ' sthyroditis, HT) and special property Myxodema (the Primary myxedema that sends out, PM), its sickness rate accounts for the first place of endocrine system disease.(thyrotropin receptor TSHR) is the major antigen that brings out AITD to thyrotropin receptor.

People TSHR molecular weight is 84.5KD, reaches 100KD after the glycosylation, and is close with the onset relation of AITD.People TSHR mainly is present on the thyroid cell film, belongs to one of guanine nucleotide regulatory protein (G albumen) coupled receptor superfamily member; By the film outskirt, stride the film district and film inner region three parts are formed: the film outskirt has 397 amino-acid residues, contains N-end, 5 glycosylation sites and two and has immunogenic peptide section.

Under physiological conditions, thyrotropic hormone (TSH) is with after TSHR combines, growth, differentiation and the function of regulating thyroid cell by adenylate cyclase AC-cAMP path and Phospholipase C-diacylglycerol and phosphoinositide path.AITD when morbidity, the body of immunologic dysfunction at TSHR produced autoantibody (Thyrotropin receptor antibody, TRAb).TRAb belongs to polyclonal antibody, act on the different loci of TSHR film outskirt (TSHRecd), difference according to its function is divided into three kinds: (1) thyroid stimulating antibody (Thyroid stimuLating antibodies, TSAb): by activated adenyl cyclase-cAMP path, the function of overstimulation thyroid follicle and growth cause hyperthyroidism; (2) Tiroidina stimulate blocker (Thyroid-stimuLating blocking antibodies, TSBAb): have retardance TSH or TSAb to thyroid excitation, suppress the growth of thyroid follicular cells, can cause that thyroid function lowers; (3) TSH in conjunction with suppress immunoglobulin (Ig) (TSH Binding inhibitorimmunoglobuLin, TBII): relevant with the outer symptom of the sick Tiroidina of Graves such as expophthalmos, pretibial myxedema.

In view of TSHR in physiological function on the thyroid follicular cells and the vital role in AITD morbidity, the TSHR purifying protein that obtains high-affinity become further investigation TSHR function and with the basis of TRAb effect.And the TSHR quantity that is present on the thyroid cell film is few, structural instability, and be difficult to purifying, and therefore people TSHR gene is carried out molecular cloning, it is significant for AITD morbidity and clinical treatment research to set up the vivoexpression system.

People TSHR gene is positioned at karyomit(e) 14q31 place, and being encoded by the opening code-reading frame of 2470bp base forms; Its cDNA in 1989 by successful clone such as Nagayama.At present, the people TSHR of prokaryotic expression can not form correct natural conformation and glycosylation, and avidity is low, uses limited.Mammalian cell can be expressed the TSHR of function, but yields poorly.Change the hTSHR gene over to TSHR film outskirt albumen that insect cell can obtain milligram quantity by baculovirus vector, but these albumen are most of water insoluble and lower with the combination rate of TSH.

Summary of the invention

The problem that the present invention solves is to provide a kind of human TSHR extracellular fusion protein and preparation method thereof, utilizes Protocols in Molecular Biology to realize the secreting, expressing of human TSHR extracellular fusion protein at Bichi yeast system, purifying and the activity of fusion rotein detected.

The present invention is achieved through the following technical solutions:

A kind of human TSHR extracellular fusion protein, this fusion rotein are to connect exogenous secreting signal peptide at the N of people's tsh receptor albumen extracellular fragment end, connect the purifying flag sequence at the C end.

Described exogenous secreting signal peptide is the α-factor factor of yeast saccharomyces cerevisiae.

Described purifying flag sequence is the flag sequence of 6 * His, by being connected with tsh receptor albumen extracellular fragment by the sequence that enteropeptidase is discerned.

Also insert c-myc antigenic determinant sequence between the sequence of described enteropeptidase identification and the 6 * His flag sequence.

Described excision N holds the human TSHR extracellular fusion protein of exogenous secreting signal peptide, and its aminoacid sequence is shown in SEQ ID NO.1.

A kind of preparation method of human TSHR extracellular fusion protein may further comprise the steps:

1) being template with the carrier that comprises people TSHR gene, is primer with primer to P, pcr amplification human TSHR extracellular gene; Described primer to P is:

Upstream primer P1:ccctcgagaa aagagggtgt tcgtctccac;

Downstream primer P2:gctctagagc atcatcatca tcttttctca ggaacttgta g;

2) the human TSHR extracellular gene with pcr amplification obtains linearizing fragment with Xho I, Xba I double digestion; And this linearizing fragment is connected with the pPICZ α A carrier of Xho I, Xba I double digestion obtains expression vector pPICZ α A-hTSHRecd;

3) competent pichia spp is arrived in expression vector pPICZ α A-hTSHRecd transfection, screening obtains the positive transformant of purpose fragment positive expression;

4) positive transformant is cultivated under 28 ℃, 250r/m condition in the BMGY substratum, when OD600 reaches 2～6, centrifugal collection thalline, resuspended with the BMMY substratum to OD ₆₀₀Be 1.0 abduction deliverings, it is to continue to cultivate 72h under 0.5%, 28 ℃, 250r/m condition that every 24h adds methyl alcohol to final concentration;

Described BMGY substratum is: add 10g yeast extract and 20g peptone in the 700ml deionized water, autoclaving adds after being cooled to room temperature: 100ml 1M phosphoric acid buffer, 100ml 10 * yeast nitrogen base, 2ml 500 * vitamin H, 100ml 10 * glycerine;

Described BMMY substratum is: add 10g yeast extract and 20g peptone in the 700ml deionized water, autoclaving adds after being cooled to room temperature: 100ml 1M phosphoric acid buffer, 100ml 10 * yeast nitrogen base, 2ml500 * vitamin H, 100ml 10 * methyl alcohol;

5) after cultivation finished, with the centrifugal 10min of recombination yeast nutrient solution room temperature 12000r/m, adding ammonium sulfate to final concentration was 50% in stirring down to get supernatant liquor; Slow magnetic agitation 4h under 4 ℃ of conditions, the centrifugal 30min of 12000r/m abandons supernatant then; The gained throw out is with 50mM, and the PBS of PH7.4 dissolving obtains the human TSHR extracellular fusion protein concentrated solution;

6) utilize the chromatography column of 6 * His flag sequence identification that the human TSHR extracellular fusion protein concentrated solution is separated, obtain the human TSHR extracellular fusion protein of purifying behind the dialysis desalination.

Described expression vector pPICZ α A-hTSHRecd includes the nucleotide sequence shown in SEQ ID NO.3.

The step that described expression vector pPICZ α A-hTSHRecd is transformed into the competence pichia spp is:

The pPICZ α A-hTSHRecd expression vector of linearization for enzyme restriction with after the competence pichia spp mixes, is added in the 0.1cm electricity revolving cup, ice-water bath 5min, in voltage 1800v, electric capacity 25uF, electric shock transforms under resistance 200 ohmic conditions;

After the electric shock, add the sorbyl alcohol that 1mL concentration is the 1M precooling immediately, mixing leaves standstill 2h under 30 ℃ of conditions, transfer to the YPDS that contains zeocin then and select to cultivate 3～7 days on the culture plate, treat that the positive transformant of laggard performing PCR evaluation and screening expressing human TSHR positive expression appears in bacterium colony.

Compared with prior art, the present invention has following beneficial technical effects:

1, be compared to TSHR (tsh receptor) quantity on the thyroid cell film few, be difficult to purifying, and prokaryotic system is expressed and baculovirus vector is low at the human TSHR extracellular protein affinity of insect cell expression, and the lower defective of combination rate of TSH; The fusion rotein that the present invention makes up is to connect exogenous secreting signal peptide at the N of people's tsh receptor albumen extracellular fragment end, makes people's tsh receptor albumen extracellular fragment enter Secretory Pathway after expressing, and makes protein obtain configuration accurately afterwards outside being secreted into born of the same parents;

What is more important can make expression vector pPICZ α A-hTSHRecd be integrated into the high efficiency stable expression that the pichia spp genome is realized this fusion rotein by electric shock; Expressed fusion protein is secreted with soluble form, has good tsh receptor antigenicity, can discern the antibody in the Graves patient serum, the monoclonal antibody TSHRmAb of TSHR;

Fusion rotein provided by the invention is a kind ofly can have the antigenic recombinant protein of tsh receptor by high efficiency stable expression, and the pichia yeast expression system that comprises pPICZ α A-hTSHRecd expression vector is a kind of good vivoexpression system;

And in the site linkage flag sequence of C end by enteropeptidase identification, purified fusion protein can be removed flag sequence by the restriction enzyme digestion reaction of enteropeptidase when being necessary easily.

2, the fusion rotein that makes up in view of the present invention has the antigenicity of TSHR, has following purposes:

The detection of the detection of a, TRAb: TRAb helps GD to differentiate mutually with other hyperthyroidism diseases; The curative effect that is used for antithyroid drug detects; The diagnosis of Thyroid-related Ophthalmopathy; GD patient's pregnancy and puerperium monitoring etc.

B, set up TSHR specific T-cells clone, preparation T cell vaccine treatment Graves hyperthyroidism.

C, immune animal are made the AITD animal model.

D, be used to prepare the TSHR monoclonal antibody specific, carry out the research of TSHR epitope.

E, in order to inducing the antibody that can discern natural TSHR, detect the expression of the outer tissue T SHR of thyroid carcinoma or Tiroidina etc.

Description of drawings

Fig. 1 is the pcr amplified fragment electrophorogram of TSHRecd gene;

Fig. 2 is the multiple clone site distribution plan of pPICZ α A carrier;

Fig. 3 is that the double digestion of expression vector pPICZ α A-hTSHRecd is identified figure;

Fig. 4 is that the direct bacterium colony PCR of positive transformant of the present invention identifies figure;

Fig. 5 is a Coomassie brilliant blue colored graph after the human TSHR extracellular fusion protein SDS-PAGE gel electrophoresis of abduction delivering;

Fig. 6 be the people TSHR fusion rotein of abduction delivering with anti-His antibody as anti-WesternBlot figure as a result;

Fig. 7 be the people TSHR fusion rotein of abduction delivering with TSHRmAb antibody as anti-Western Blot figure as a result.

Embodiment

The invention provides a kind of can efficiently expressing and have the antigenic recombinant protein of TSHR, but, in its good vivoexpression system, obtain the solubility recombinant protein with the antigenic secreted form of TSHR of high expression level by setting up the Pichia yeast engineering of secreting, expressing people TSHR.Below the present invention is elaborated, the explanation of the invention is not limited.

1, the structure of expression vector pPICZ α A-hTSHRecd:

The clone of a, hTSHRecd extracellular fragment gene

According to people TSHR gene order (NM_000369.2) among the GenBank, design and synthesize the oligonucleotide Auele Specific Primer of a pair of hTSHRecd cDNA coding region that is used to increase:

Upstream primer P1:cc CtcgagAa aagagggtgt tcgtctccac 30;

Downstream primer P2:gct CtagagC atcatcatca tcttttctca ggaacttgta g 41;

P1 contains Xho I restriction enzyme site (line part), and P2 contains the sequence of Xba I restriction enzyme site (line part) and enteropeptidase identification.

Right with P1, P2 as primer, be template with pcDNA3.1-hTSHR plasmid (being so kind as to give length 7700bp) by Chen professor Chun-Rong of medical college of California, USA university, the people TSHR gene of pcr amplification 1227bp;

The PCR reaction conditions is: 94 ℃ of pre-sex change 120s, 94 ℃ of sex change 60s, 58 ℃ of annealing 60s, 72 ℃ of extension 120s, 35 circulations; 72 ℃ are extended 5min eventually;

After pcr amplification was finished, the agarose gel electrophoresis with 1% was identified the PCR product:

Draw PCR product 5 μ L with micropipet,, slowly add on the gel in the sample hole with the abundant mixing of 1 μ L, 6 * sample-loading buffer; With DL1000DNA Marker is molecular weight standard, last sample 4 μ L; 100V voltage, electrophoresis 30 minutes;

Show that through Bio-Rad Gel Doc XR gel imaging analysis system electrophoretic band is clear, the result can see the band of 1.2Kb clearly as shown in Figure 1, and the PCR fragment that amplifies is consistent with the intended purposes clip size; PCR product band carries out dna sequencing to the PCR product after fragment conforms to expection, and the sequencing result of hTSHRecd extracellular fragment gene is shown in SEQ ID NO.2;

Use DNA purification kit purified pcr product, obtain the hTSHRecd extracellular fragment gene of purifying.

The double digestion of b, hTSHRecd extracellular fragment gene

With hTSHRecd extracellular fragment gene Xho I, the Xba I double digestion of purifying, the enzyme system of cutting of 20 μ L is:

The Xba I restriction enzyme of the hTSHRecd extracellular fragment gene of 5 μ L, the Xho I restriction enzyme of 1 μ L, 1 μ L, 10 * M buffer of 2 μ L, the sterilization deionized water is supplied 20 μ L; 37 ℃ of water-baths, enzyme is cut 2h;

HTSHRecd extracellular fragment gene behind the double digestion is separated the electrophoretic band of the hTSHRecd extracellular fragment gene that comprises with adsorption column CA1 purifying, centrifugal collection with the low melting-point agarose gel electrophoresis; HTSHRecd extracellular fragment gene for the band cohesive terminus that obtains detects its purity with Nanodrop SpectrophotometerNd-1000, and OD260/OD280 ratio is 1.8.

The double digestion of c, pPICZ α A carrier

With vector plasmid pPICZ α A (its multiple clone site collection of illustrative plates as shown in Figure 2) transformed competence colibacillus TOP10F ' bacterium, picking mono-clonal colony inoculation was in the LB nutrient solution that contains penbritin (Amp) 100mg/L after 37 ℃ of thermostat containers were cultivated 12h, 37 ℃, collect bacterium after the 160rpm overnight incubation and extract plasmid pPICZ α A;

The plasmid pPICZ α A that collects is obtained the linearizing fragment through Xho I, Xba I double digestion, reclaim test kit with DNA and reclaim.

Connection and the extraction of d, expression vector pPICZ α A-hTSHRecd

The linearizing hTSHRecd extracellular fragment gene fragment of same Xho I, Xba I double digestion is connected with the T4DNA ligase enzyme with pPICZ α A carrier, obtains expression vector pPICZ α A-hTSHRecd;

With recombinant expression vector pPICZ α A-hTSHRecd transformed competence colibacillus DH5 α bacterium, picking mono-clonal colony inoculation was in the LB nutrient solution that contains penbritin (Amp) 100mg/L after 37 ℃ of thermostat containers were cultivated 12h, collect bacterium after 37 ℃ of cultivations of shaking table, use no intracellular toxin plasmid and extract test kit extraction recombinant plasmid in a large number, Nanodrop Spectrophotometer Nd-1000 detects its purity, and OD260/OD280 ratio is 1.86;

Application limitations restriction endonuclease Pme I enzyme is cut (restriction enzyme site in the pPICZ α A carrier) recombinant plasmid and is made it linearizing, uses ethanol precipitation and concentrates linearizing recombinant plasmid, makes it concentration and reaches 1 μ g/ μ L ,-20 ℃ of preservations.

Agarose gel electrophoresis shows the about 4800bp of length of recombinant plasmid, agarose gel electrophoresis behind the recombinant plasmid Xho I that collects, the Xba I double digestion is identified, as shown in Figure 3: wherein swimming lane 1 sees that at 3600bp, 1200bp place (wherein the 3600bp fragment is carrier pPICZ α A to two electrophoretic bands, the 1200bp fragment is the encoding gene of hTSHRecd), M is the DNA ladder of 200bp; Illustrate that recombinant plasmid obtains the insertion fragment of expection size after enzyme is cut;

Send Beijing AudioCodes Bioisystech Co., Ltd to carry out dna sequencing recombinant plasmid, the result shows that the α-factor factor (α-mating factor) of the yeast saccharomyces cerevisiae on goal gene TSHR and the pPICZ α A carrier correctly is connected, form new fusion gene, its nucleotide sequence is specifically shown in SEQ ID NO.3;

α-bootable recombinant protein of the factor factor enters Secretory Pathway, makes protein obtain configuration accurately afterwards outside being secreted into born of the same parents.

2, the Construction of eukaryotic of human TSHR extracellular fusion protein

Because pichia yeast expression system is fairly perfect heterologous gene expression system, its expression alien gene has the following advantages: the distinctive alcohol oxidase gene promoter of expression vector (AOX1) has strong inducibility and strong startability, be suitable for the high-level abduction delivering of foreign gene, the high expression level amount of having reported is respectively 22g/L (in the born of the same parents) and 14.8g/L (secretion); Expression product can glycosylation and is belonged to secretion type expression, helps proteic separation and purifying, and the cultivation of can high-density continuously fermenting, and can adapt to need of industrial production; Substratum is inexpensive; Genetic background is clear, is convenient to carry out genetic manipulation; Foreign gene is stable existence in host cell.50%～75% pichia yeast expression system can be expressed the interested any extrinsic protein of people with reasonable levels; Some baculovirus expression system can not expressed protein but can successful expression at pichia yeast expression system.Therefore, the present invention adopts pichia spp as host cell.

The foundation of the pichia spp highly effective eukaryon expression system of TSHRecd is specially:

The separation and purification of a, yeast strain:

Inoculation yeast bacterium GS115 (adds the 10g yeast extract in the YPD of 5mL in the 900ml deionized water, autoclaving behind the 20g peptone, add 10 * D glucose 100ml after being cooled to room temperature, deposit for 4 ℃) liquid nutrient medium, 30 ℃, the 200rpm shaken overnight, coating YPD flat board, cultivate 48h for 30 ℃, do the dibbling separation and purification with YNB minimum medium MD is dull and stereotyped with the supplemental medium MDH flat board that contains Histidine, select single bacterium colony of on minimum medium, not growing, the GS115 Histidine auxotrophy strain that separation and purification goes out, 4 ℃ of preservations in growth on the supplemental medium.

The preparation of b, GS115 competent cell:

Picking through the single colony inoculation of the GS115 of separation and purification Histidine auxotrophy strain in the YPD of 10mL substratum, 30 ℃ of shaken overnight;

Be seeded to seed liquor in the fresh 200mLYPD substratum with 1: 100 next day, and 30 ℃, the 200rpm incubated overnight is to OD600=1.3;

Bacterium liquid is in 4 ℃, and centrifugal 5 minutes of 1500 * g abandons supernatant, and precipitation is resuspended in the sterilization ddH of 200mL ice precooling ₂Among the O; In 4 ℃, 1500 * g recentrifuge 5 minutes is abandoned supernatant, and precipitation is resuspended in the sterilization ddH of 100mL ice precooling ₂Among the O.

Carry out 2 centrifugations precipitations then respectively, be resuspended in successively in the 1M sorbyl alcohol liquid of ice precooling of 1M of 20mL, 1mL ice precooling, the about 1.5mL of final volume obtains the GS115 competent cell.

C, recombinant expression vector electricity transform pichia spp GS115 competent cell:

Get 80 μ L yeast competent cells respectively and mix through linearizing recombinant expression plasmid of restriction endonuclease Pme I or vector plasmid with 10 μ g, with sample injector sample drop is added in the 0.1cm electricity revolving cup, ice-water bath 5min uses Bio-Rad Gene

The II electroporation is in voltage 1800v, electric capacity 25uF, and electricity transforms under resistance 200 ohmic conditions;

After the electric shock, adding 1mL concentration immediately is the sorbyl alcohol of 1M ice precooling, and mixing is transferred in the 15mL centrifuge tube, 30 ℃, leaves standstill 2h; Get respectively 10,25,50,100 and 200uL bacterium liquid coat on the YPDS flat board that contains 100 μ g/mL Zeocin (belonging to bleomycin family), hatched 3-10 days for 30 ℃, until bacterium colony occurring.

The screening of d, pichia spp positive transformant and evaluation

With well-grown mono-clonal bacterium colony on the YPDS flat board that is containing 100 μ g/mL Zeocin through autoclaved toothpick picking, be inoculated in the YPD substratum in 30 ℃, 200rpm, frozen after the incubated overnight in-80 ℃;

The remainder of same bacterium colony is chosen in the 200 μ L EP pipes of corresponding numbering and (added 12.5 μ L sterilization deionized water), adding 2.5 μ L concentration is the lysozyme soln of 10U/ μ L, mixing, in 30 ℃ put into after leaving standstill 10 minutes-80 ℃ placed 10 minutes direct bacterium colony pcr template, carry out the pcr amplification evaluation with 5 ' AOX1 primer P3,3 ' AOX1 primer P4;

P3：gactggttcc?aattgacaag?c?21；

P4：gcaaatggca?ttctgacatc?c?21；

Direct bacterium colony PCR system:

5 * primeSTARTM Buffer (has added Mg ²⁺) 10uL;

DNTP Mixture (each 2.5mM) 4uL;

5′AOX1?primer(10pmol/μl) 1uL；

3′AOX1?primer(10pmol/μl) 1uL；

Pcr template 5uL;

The sterilization deionized water is mended to 49.5uL;

Behind the mixing 95 ℃, hatched 5 minutes; Add PrimeSTARTMHS DNA Polymerase0.5uL, mixing;

The PCR reaction conditions is: 94 ℃ of sex change 60s, and 58 ℃ of annealing 60s, 72 ℃ are extended 60s, and after 30 circulations, 72 ℃ are extended 7min eventually.

Through agarose gel electrophoresis qualification result such as Fig. 4: swimming lane 1 visual length is about the electrophoretic band of 1800bp, conforms to the expection fragment length (588bp+1227bp, 588bp are the length of AOX1, and 1227 is target fragment length).

3, the abduction delivering of fusion rotein and purifying

The inoculation positive transformant is in 10mL BMGY substratum, and 28 ℃, 250rpm overnight incubation are inoculated positive control GS115/His+MutS Albumin simultaneously, and the empty bacterium of negative control pPICZ α A transformant and GS115 is cultivated, and works as OD ₆₀₀Reach at 2～6 o'clock, room temperature, the centrifugal collection of 1500rpm bacterium, resuspended with the 50mLBMMY substratum to OD ₆₀₀Be that 1.0 continuation are at 28 ℃, 250rpm inducing culture (can start the expression of AOX1 inducible protein thereby contain methyl alcohol among the BMMY), collect each 1.4mL of bacterium liquid respectively at 0h, 6h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, supply the BMMY substratum to 50mL, it is 0.5% that every 24h adds methyl alcohol to final concentration; With adding proteinase inhibitor cocktail 20 μ L in the supernatant of the centrifugal back of sample, in-80 ℃ of preservations.

For the recombination yeast nutrient solution be further purified for:

Centrifugal 10 minutes of room temperature 12000rpm, adding saturated ammonium sulphate to final concentration is slow magnetic agitation 4h under 50%, 4 ℃ of condition in stirring down to get supernatant liquor, the centrifugal 30min of 12000rpm abandons supernatant then; The gained throw out is with 50mM, and the PBS of PH7.4 dissolving obtains the human TSHR extracellular fusion protein concentrated solution;

Utilize the chromatography column of 6 * His flag sequence identification that the human TSHR extracellular fusion protein concentrated solution is separated, the present invention specifically uses

Purification Kit (Cat.No.635515) comes purifying, carries out the purifying of human TSHR extracellular fusion protein according to its specification sheets.

4, the evaluation of the fusion rotein of Biao Daing

The Coomassie brilliant blue dyeing of a, fusion rotein

Get the 1.4mL yeast and induce supernatant to be sub-packed in two 1.5mlEP pipes, add the mixed mixing of 0.7mL20% trichoroacetic acid(TCA) solution, place 30min on ice, 4 ℃, the centrifugal 20min of 12000rpm; Protein precipitation is with the washing 3 times of vibrating repeatedly of 80% ethanol of 0.8mL ice precooling, and 4 ℃, the centrifugal 5min of 12000rpm removes supernatant, the natural air drying precipitation;

With 40 μ L μ L sterilization ddH ₂The resuspended protein precipitation of O is got 24 μ L and is added 5 * electrophoretic buffer, 6 μ L, goes up sample behind 100 ℃ of sex change 10min and carries out the SDS-PAGE gel electrophoresis, deposition condition: step 1:80V, 30 minutes; Step 2:120V treats that tetrabromophenol sulfonphthalein runs out of sheet glass and can cut off the power supply;

After taking out, the SDS-PAGE gel carries out Coomassie brilliant blue dyeing, Figure 5 shows that and be labeled as No. 149 transformant culture supernatant Coomassie brilliant blue coloration result after trichoroacetic acid(TCA) concentrates the capable SDS-PAGE gel electrophoresis in back: wherein, M is Maker, swimming lane 1～9 is respectively 96h, 84h, 72h, 60h, 48h, 36h, 24h, 12h, 6h, 0h culture supernatant enriched product, Bio-Rad gel imaging analysis system shows that swimming lane 1-7 all can see the set goal albumen at 47KD band place, and promptly No. 149 transformants promptly have a small amount of protein expression in cultivating 12h; After inducing 72h, it is maximum that the target protein expression amount reaches.In many strains positive transformant that the applicant makes up, most of bacterial strain has target protein to express after inducing 36h.

The Western Blot of b, expression product identifies

Yeast induces supernatant behind the concentrating of step a, electrophoresis, excision separation gel and unnecessary part; Take off gel, the protein electricity is transferred on the NC film, carrying out Western Blot identifies, wherein one anti-be Anti-His, TSHRmAb (professor ChenChun-Rong of medical college of California, USA university is so kind as to give) with 1: 200 dilution proportion of TBST, two anti-be goat-anti people or sheep anti-mouse igg with the horseradish peroxidase-labeled of TBST ratio preparation in 1: 5000.The molecular weight of Bio-Rad Gel Doc XR gel imaging analysis systems analysis target stripe and clean optical density value;

With Anti-His as shown in Figure 6 as a Western Blot result who resists, wherein, swimming lane M is albumen Maker, and

swimming lane

1,2,3 is respectively the different positive transformant inductive human TSHR extracellular fusion protein of numbering, and it all can see clear band at the target sizes place;

With TSHRmAb as shown in Figure 7 as a Western Blot result who resists, wherein, swimming lane M is albumen Maker, swimming lane 1 is GS115/His+MutS Albumin, swimming lane 2 is the GS115 tropina, swimming lane 3 is a pPICZ α A transformant tropina, and

swimming lane

4,5,6 is respectively the different positive transformant inductive human TSHR extracellular fusion protein of numbering;

Swimming lane

1,2,3 does not demonstrate corresponding band in contrast, and

swimming lane

4,5,6 all can be seen clear band at the target sizes place;

More than two groups of Western Blot detect explanation, the people TSHR fusion rotein that the present invention makes up has the antigenicity of TSHR and the characteristic of His purification tag.

5, indirect ELSIA method is measured the functional examination of fusion rotein

Centrifugal 10 minutes of recombination yeast nutrient solution room temperature 12000rpm, dropwise to add saturated ammonium sulphate to ammonium sulfate final concentration down be 50% in stirring to get supernatant liquor, 4 ℃, slow magnetic agitation 4h, it is fully precipitated, and the centrifugal 30min of 12000rpm abandons supernatant, the gained throw out is with 50mM, and the PBS of PH7.4 dissolves;

The good fusion rotein solution of dissolving is packed in the suitable dialysis card of the molecular weight of anticipating, with PBS (50mM, the PH7.4) desalination of fully dialysing, slow magnetic agitation, every 4h changes liquid once, it is yellow for not having to survey extracellular fluid dialysis to Nai Shi reagent;

Take out the dialysis card, it is embedded in the Macrogol 2000 0, extremely moisture is blotted fully in the dialysis card, then coating buffer (the 0.015MNa that adding prepares in the dialysis card ₂CO ₃, 0.035MNaHCO ₃, 0.02%NaN ₃, pH:9.6), the concentration that makes fusion rotein is 10 μ g/ml, every hole adds 100 μ l bag by 96 orifice plates; Resist as one with Graves patient positive serum, TSHRmAb respectively and add in the different holes, the goat-anti people or the sheep anti-mouse igg that add horseradish peroxidase-labeled in the respective aperture, simultaneously with of the contrast of normal people's pooled serum group as Graves patient positive serum group, with of the contrast of PBS group as the TSHRmAb group, carrying out ELSIA detects, the result is as shown in table 1, TSHRmAb group and PBS group, GD patient's pooled serum group and normal people's pooled serum group OD ₄₅₀There is significant difference (P＜0.05) in value, shows that fusion rotein has good antigenic activity.

The ELISA of table 1 human TSHR extracellular fusion protein antigenic activity detects

Group	OD ₄₅₀Value (x ± s)
		The TSHRmAb group	0.57±0.11*
The PBS group	0.13±0.03
		GD patient's pooled serum group	3.08±0.01**
Normal people's pooled serum group	0.88±0.03

* P＜0.05vs PBS organizes; * P＜0.05vs normal people's pooled serum group

6, determine the sequence of human TSHR extracellular fusion protein

Check order through biotech firm for above-mentioned aminoacid sequence with the antigenic fusion rotein of tsh receptor, concrete aminoacid sequence is shown in SEQ ID NO.1, wherein, the 397th～401 amino-acid residue is the enteropeptidase restriction enzyme site, and the 402nd～418 amino-acid residue is the c-myc antigenic determinant; The 419th～424 amino-acid residue is 6 * His tail.Using enteropeptidase carries out enzyme and cuts and can obtain the tsh receptor albumen that length is 396 amino acid lengths; And that the Mfa factor can be worn in the membrane process at fusion rotein as signal peptide is cut, so the hTSHRecd fusion rotein in culture supernatant does not contain the Mfa factor.

Sequence table

The nucleotides sequence tabulation

＜110〉No.1 Hospital Attached to Medical College, Xi'an Communication Univ

＜120〉a kind of human TSHR extracellular fusion protein and preparation method thereof

<160>3

<210>1

<211>424

<212>PRT

＜213〉synthetic

<400>1

Gly?Cys?Ser?Ser?Pro?Pro?Cys?Glu?Cys?His?Gln?Glu?Glu?Asp?Phe?Arg?Val?Thr?Cys?Lys

1 5 10 15 20

Asp?Ile?Gln?Arg?IIe?Pro?Ser?Leu?Pro?Pro?Ser?Thr?Gln?Thr?Leu?Lys?Leu?Ile?Glu?Thr

25 30 35 40

His?Leu?Arg?Thr?Ile?Pro?Ser?His?Ala?Phe?Ser?Asn?Leu?Pro?Asn?Ile?Ser?Arg?Ile?Tyr

45 50 55 60

Val?Ser?Ile?Asp?Val?Thr?Leu?Gln?Gln?Leu?Glu?Ser?His?Ser?Phe?Tyr?Asn?Leu?Ser?Lys

65 70 75 80

Val?Thr?His?Ile?Glu?Ile?Arg?Asn?Thr?Arg?Asn?Leu?Thr?Tyr?Ile?Asp?Pro?Asp?Ala?Leu

85 90 95 100

Lys?Glu?Leu?Pro?Leu?Leu?Lys?Phe?Leu?Gly?Ile?Phe?Asn?Thr?Gly?Leu?Lys?Met?Phe?Pro

105 110 115 120

Asp?Leu?Thr?Lys?Val?Tyr?Ser?Thr?Asp?Ile?Phe?Phe?Ile?Leu?Glu?Ile?Thr?Asp?Asn?Pro

125 130 135 140

Tyr?Met?Thr?Ser?Ile?Pro?Val?Asn?Ala?Phe?Gln?Gly?Leu?Cys?Asn?Glu?Thr?Leu?Thr?Leu

145 150 155 160

Lys?Leu?Tyr?Asn?Asn?Gly?Phe?Thr?Ser?Val?Gln?Gly?Tyr?Ala?Phe?Asn?Gly?Thr?Lys?Leu

165 170 175 180

Asp?Ala?Val?Tyr?Leu?Asn?Lys?Asn?Lys?Tyr?Leu?Thr?Val?Ile?Asp?Lys?Asp?Ala?Phe?Gly

185 190 195 200

Gly?Val?Tyr?Ser?Gly?Pro?Ser?Leu?Leu?Asp?Val?Ser?Gln?Thr?Ser?Val?Thr?Ala?Leu?Pro

205 210 215 220

Ser?Lys?Gly?Leu?Glu?His?Leu?Lys?Glu?Leu?Ile?Ala?Arg?Asn?Thr?Trp?Thr?Leu?Lys?Lys

225 230 235 240

Leu?Pro?Leu?Ser?Leu?Ser?Phe?Leu?His?Leu?Thr?Arg?Ala?Asp?Leu?Ser?Tyr?Pro?Ser?His

245 250 255 260

Cys?Cys?Ala?Phe?Lys?Asn?Gln?Lys?Lys?Ile?Arg?Gly?Ile?Leu?Glu?Ser?Leu?Met?Cys?Asn

265 270 275 280

Glu?Ser?Ser?Met?Gln?Ser?Leu?Arg?Gln?Arg?Lys?Ser?Val?Asn?Ala?Leu?Asn?Ser?Pro?Leu

285 290 295 300

His?Gln?Glu?Tyr?Glu?Glu?Asn?Leu?Gly?Asp?Ser?Ile?Val?Gly?Tyr?Lys?Glu?Lys?Ser?Lys

305 310 315 320

Phe?Gln?Asp?Thr?His?Asn?Asn?Ala?His?Tyr?Tyr?Val?Phe?Phe?Glu?Glu?Gln?Glu?Asp?Glu

325 330 335 340

Ile?Ile?Gly?Phe?Gly?Gln?Glu?Leu?Lys?Asn?Pro?Gln?Glu?Glu?Thr?Leu?Gln?Ala?Phe?Asp

345 350 355 360

Ser?His?Tyr?Asp?Tyr?Thr?Ile?Cys?Gly?Asp?Ser?Glu?Asp?Met?Val?Cys?Thr?Pro?Lys?Ser

365 370 375 380

Asp?Glu?Phe?Asn?Pro?Cys?Glu?Asp?Ile?Met?Gly?Tyr?Lys?Phe?Leu?Arg?Lys?Asp?Asp?Asp

385 390 395 400

Asp?Ala?Leu?Glu?Gln?Lys?Leu?Ile?Ser?Glu?Glu?Asp?Leu?Asn?Ser?Ala?Val?Asp?His?His

405 410 415 420

His?His?His?His

424

<210>2

<211>1227

<212>DNA

＜213〉synthetic

<400>2

ccctcgagaa?aagagggtgt?tcgtctccac?cctgcgagtg?ccatcaggag?gaggacttca 60

gagtcacctg?caaggatatt?caacgcatcc?ccagcttacc?gcccagtacg?cagactctga 120

agcttattga?gactcacctg?agaactattc?caagtcatgc?attttctaat?ctgcccaata 180

tttccagaat?ctacgtatct?atagatgtga?ctctgcagca?gctggaatca?cactccttct 240

acaatttgag?taaagtgact?cacatagaaa?ttcggaatac?caggaactta?acttacatag 300

accctgatgc?cctcaaagag?ctccccctcc?taaagttcct?tggcattttc?aacactggac 360

ttaaaatgtt?ccctgacctg?accaaagttt?attccactga?tatattcttt?atacttgaaa 420

ttacagacaa?cccttacatg?acgtcaatcc?ctgtgaacgc?gtttcaggga?ctatgcaatg 480

aaaccttgac?actgaagctg?tacaacaatg?gctttacttc?agtccaagga?tatgctttca 540

atgggacaaa?gctggatgct?gtttacctaa?acaagaataa?atacctgaca?gttattgaca 600

aagatgcatt?tggaggagta?tacagtggac?caagcttgct?ggacgtgtct?caaaccagtg 660

tcactgccct?tccatccaaa?ggcctggagc?acctgaagga?actgatagca?agaaacacct 720

ggactcttaa?gaaacttcca?ctttccttga?gtttccttca?cctcacacgg?gctgaccttt 780

cttacccaag?ccactgctgt?gcttttaaga?atcagaagaa?aatcagagga?atccttgagt 840

ccttgatgtg?taatgagagc?agtatgcaga?gcttgcgcca?gagaaaatct?gtgaatgcct 900

tgaatagccc?cctccaccag?gaatatgaag?agaatctggg?tgacagcatt?gttgggtaca 960

aggaaaagtc?caagttccag?gatactcata?acaacgctca?ttattacgtc?ttctttgaag 1020

aacaagagga?tgatatcatt?ggttttggcc?aggagctcaa?aaacccccag?gaagagactc 1080

tacaagcttt?tgacagccat?tatgactaca?ccatatgtgg?ggacagtgaa?gacatggtgt 1140

gtacccccaa?gtccgatgag?ttcaacccgt?gtgaagacat?aatgggctac?aagttcctga 1200

gaaaagatga?tgatgatgct?ctagagc 1227

<210>3

<211>1708

<212>DNA

＜213〉synthetic

<400>3

gcaagaccgg?tcttctcgta?agtgcccaac?ttgaactgag?gaacagtcat?gtctaaggct 60

acaaactcaa?tgatgatgat?gatgatggtc?gacggcgcta?ttcagatcct?cttctgagat 120

gagtttttgt?tctagagcat?catcatcatc?ttttctcagg?aacttgtagc?ccattatgtc 180

ttcacacggg?ttgaactcat?cggacttggg?ggtacacacc?atgtcttcac?tgtccccaca 240

tatggtgtag?tcataatggc?tgtcaaaagc?ttgtagagtc?tcttcctggg?ggtttttgag 300

ctcctggcca?aaaccaatga?tatcatcctc?ttgttcttca?aagaagacgt?aataatgagc 360

gttgttatga?gtatcctgga?acttggactt?ttccttgtac?ccaacaatgc?tgtcacccag 420

attctcttca?tattcctggt?ggagggggct?attcaaggca?ttcacagatt?ttctctggcg 480

caagctctgc?atactgctct?cattacacat?caaggactca?aggattcctc?tgattttctt 540

ctgattctta?aaagcacagc?agtggcttgg?gtaagaaagg?tcagcccgtg?tgaggtgaag 600

gaaactcaag?gaaagtggaa?gtttcttaag?agtccaggtg?tttcttgcta?tcagttcctt 660

caggtgctcc?aggcctttgg?atggaagggc?agtgacactg?gtttgagaca?cgtccagcaa 720

gcttggtcca?ctgtatactc?ctccaaatgc?atctttgtca?ataactgtca?ggtatttatt 780

cttgtttagg?taaacagcat?ccagctttgt?cccattgaaa?gcatatcctt?ggactgaagt 840

aaagccattg?ttgtacagct?tcagtgtcaa?ggtttcattg?catagtccct?gaaacgcgtt 900

cacagggatt?gacgtcatgt?aagggttgtc?tgtaatttca?agtataaaga?atatatcagt 960

ggaataaact?ttggtcaggt?cagggaacat?tttaagtcca?gtgttgaaaa?tgccaaggaa 1020

ctttaggagg?gggagctctt?tgagggcatc?agggtctatg?taagttaagt?tcctggtatt 1080

ccgaatttct?atgtgagtca?ctttactcaa?attgtagaag?gagtgtgatt?ccagctgctg 1140

cagagtcaca?tctatagata?cgtagattct?ggaaatattg?ggcagattag?aaaatgcatg 1200

acttggaata?gttctcaggt?gagtctcaat?aagcttcaga?gtctgcgtac?tgggcggtaa 1260

gctggggatg?cgttgaatat?ccttgcaggt?gactctgaag?tcctcctcct?gatggcactc 1320

gcagggtgga?gacgaacacc?ctcttttctc?gaggtaccga?tccgagacgg?ccggctgggc 1380

cacgtgaatt?cagcttcagc?ctctcttttc?tcgagagata?ccccttcttc?tttagcagca 1440

atgctggcaa?tagtagtatt?tataaacaat?aacccgttat?ttgtgctgtt?ggaaaatggc 1500

aaaacagcaa?catcgaaatc?cccttctaaa?tctgagtaac?cgatgacagc?ttcagccgga 1560

atttgtgccg?tttcatcttc?tgttgtagtg?ttgactggag?cagctaatgc?ggaggatgct 1620

gcgaataaaa?cagcagtaaa?aattgaagga?aatctcatcg?tttcgaataa?ttagttgttt 1680

tttgatcttc?tcaagttgtc?gtaaaagt 1708

Claims

1. human TSHR extracellular fusion protein is characterized in that: this fusion rotein is to connect exogenous secreting signal peptide at the N of people's tsh receptor albumen extracellular fragment end, connects the purifying flag sequence at the C end.

2. human TSHR extracellular fusion protein as claimed in claim 1 is characterized in that: described exogenous secreting signal peptide is the α-factor factor of yeast saccharomyces cerevisiae.

3. human TSHR extracellular fusion protein as claimed in claim 2 is characterized in that: described purifying flag sequence is the flag sequence of 6 * His, by being connected with tsh receptor albumen extracellular fragment by the sequence that enteropeptidase is discerned.

4. human TSHR extracellular fusion protein as claimed in claim 3 is characterized in that: also insert c-myc antigenic determinant sequence between the sequence of enteropeptidase identification and 6 * His flag sequence.

5. human TSHR extracellular fusion protein as claimed in claim 4 is characterized in that: excision N holds the human TSHR extracellular fusion protein of exogenous secreting signal peptide, and its aminoacid sequence is shown in SEQ IDNO.1.

6. the preparation method of a human TSHR extracellular fusion protein is characterized in that, may further comprise the steps:

Upstream primer P1:ccctcgagaa aagagggtgt tcgtctccac;

Downstream primer P2:gctctagagc atcatcatca tcttttctca ggaacttgta g;

7. the preparation method of human TSHR extracellular fusion protein as claimed in claim 6, it is characterized in that: described expression vector pPICZ α A-hTSHRecd includes the nucleotide sequence shown in SEQ ID NO.3.

8. the preparation method of human TSHR extracellular fusion protein as claimed in claim 6 is characterized in that, expression vector pPICZ α A-hTSHRecd transfection competence pichia spp is:

The pPICZ α A-hTSHRecd expression vector of linearization for enzyme restriction with after the competence pichia spp mixes, is dripped in 0.1cm electricity revolving cup, ice-water bath 5min, in voltage 1800V, electric capacity 25uF, electric shock transforms under resistance 200 ohmic conditions;

After the electric shock, add the sorbyl alcohol that 1mL concentration is the 1M precooling immediately, mixing, leave standstill 2h under 30 ℃ of conditions, transfer to the YPDS that contains zeocin then and select to cultivate 3～7d on the culture plate, treat to carry out after bacterium colony occurs the positive transformant that bacterium colony PCR identifies, human TSHR extracellular fusion protein is expressed in western blot screening.